scholarly journals OPTIMIZATION OF MOLECULAR DETECTION FOR Vibrio cholerae and PATHOGENIC Escherichia coli USING MULTIPLEX PCR

2021 ◽  
pp. e299
Author(s):  
Diana Elizabeth Waturangi ◽  
Jason Petrus ◽  
Rico Kosasih ◽  
Felicia Roseline
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Vibrio Cholerae ◽  
False Positive ◽  
Rapid Detection ◽  
Pathogenic Bacteria ◽  
Molecular Detection ◽  
False Negative ◽  
E Coli ◽  
Pathogenic Escherichia Coli

Vibrio cholerae and pathogenic Escherichia coli were considered as main causative agent foodborne diseases especially in many developing countries, such as Indonesia. Thereby, rapid detection of these pathogenic bacteria is necessary to treat food-borne related diseases causing by these bacteria. In this case, multiplex PCR allows multiple genes amplification in one reaction thereby enable to perform rapid detection of these pathogenic bacteria. The objective of this study is to optimize uniplex and multiples PCR of V. cholerae and pathogenic E. coli detection and determine the sensitivity and specificity of this assays. We used various virulence genes for each pathogenic bacterium as markers for uniplex and multiplex PCR detection. Based on this research, the optimum results of V. cholerae and pathogenic E. coli were obtained with a primer concentration of 16 µM for ctxA and ompU, 30 µM for ace, and 50 µM for zot, and toxR; 2 µM for elt and 5 µM for stx, respectively. Finally, based on the standardization method by ISO/TS 20836 these assays had 0% false positive, 0% false negative, 100% specificity, and 100% sensitivity; 0% false positive, 4% false negative, 100% specificity, and 96% sensitivity for V. cholerae and pathogenic E. coli respectively. The optimized method was qualified to be used as a molecular detection for V. cholerae as well as EHEC and ETEC detection according to ISO/TS 20836 (2017)  from drinking water samples.

scholarly journals Development of Molecular Rapid Detection for Vibrio cholerae and Escherichia coli

2020 ◽  
Author(s):  
Diana Elizabeth Waturangi ◽  
JASON PETRUS ◽  
RICO KOSASIH ◽  
GLORIA RAISSA
Keyword(s):  
Escherichia Coli ◽  
Vibrio Cholerae ◽  
False Positive ◽  
Rapid Detection ◽  
Pathogenic Bacteria ◽  
Molecular Detection ◽  
Detection Method ◽  
False Negative ◽  
E Coli ◽  
Drinking Water Samples

Abstract Background: Vibrio cholerae and Escherichia coli were main causative agent foodborne diseases, especially in many developing countries, such as Indonesia. Thereby, rapid detection of these pathogenic bacteria is necessary to quickly detect infection that occurred so it can be treated immediately. In this case, multiplex PCR allows multiple genes amplification in one reaction thereby enable to perform rapid detection of these pathogenic bacteria. The objective of this study is to develop rapid molecular detection of V. Cholerae and E. coli and analyze the sensitivity and specificity of this assay.Result: In this study, we used various virulence genes in each pathogenic bacteria as marker to develop rapid molecular detection. Based on this research, optimum results of V. cholerae and E. coli rapid detection were obtained with a primer concentration of 16 µM for ctxA and ompU, 30 µM for ace, and 50 µM for zot, and toxR; 2 µM for elt and 5 µM for stx, respectively. Finally, based on the method standardization by ISO/TS 20836 these assays had 0% false positive, 0% false negative, 100% specificity, and 100% sensitivity; 0% false positive, 4% false negative, 100% specificity, and 96% sensitivity for V. cholerae and E. coli respectively. Conclusion: The optimized method was qualified to be used as a detection method for V. cholerae and E. coli detection according to ISO/TS 20836 (2017) and EHEC and ETEC contamination in drinking water samples.

scholarly journals Molecular Detection and Identification of Intimin Alleles in Pathogenic Escherichia coli by Multiplex PCR

1999 ◽  
Vol 37 (8) ◽  
pp. 2719-2722 ◽  
Author(s):  
Sean D. Reid ◽  
David J. Betting ◽  
Thomas S. Whittam
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Molecular Detection ◽  
Pcr Assay ◽  
E Coli ◽  
Detection And Identification ◽  
Pathogenic Escherichia Coli

A multiplex PCR was designed to detect the eae gene and simultaneously identify specific alleles in pathogenicEscherichia coli. The method was tested on 87 strains representing the diarrheagenic E. coli clones. The results show that the PCR assay accurately detects eae and resolves alleles encoding the α, β, and γ intimin variants.

IDENTIFICATION OF THE AVIAN PATHOTYPE OF ESCHERICHIA COLI ON LAYER FLOCKS BY MULTIPLEX PCR

2019 ◽  
Vol 7 ◽  
pp. 905-911
Author(s):  
Marilena Burtan ◽  
Virgilia Popa ◽  
Maria Rodica Gurau ◽  
Doina Danes
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Virulence Genes ◽  
Virulence Gene ◽  
Economic Losses ◽  
Ompa Gene ◽  
Avian Pathogenic Escherichia Coli ◽  
E Coli ◽  
Pathogenic Escherichia Coli

Introduction: Colibacillosis in poultry is determined by avian pathogenic Escherichia coli (APEC) and represents an important source of economic losses  in the poultry industry. APEC’s pathogenicity relies on the presence and expression of different virulence factors. The genes ompA , iss  and  fimH, encoding the outer membrane protein, the protein inducing resistance to complement and the synthesis of type 1 fimbria are present in APEC strains. Objective: Escherichia coli strains isolated from layers were analysed to assess the pathotype they belong to. Methods: In order to detect the three genes associated with APEC strains, 16 E. coli isolates were investigated for virulence associated genes ompA, iss and fimH, using multiplex PCR. Results: From the 16 E.coli strains submitted, multiplex PCR assessment revealed that 14 (87.5%) of the E. coli strains isolated contained at least one virulence gene, while 2 (12.5%) strains did not harbour any of the virulence genes tested. The fimH gene was noted in 13 (81.25%) of the strains tested, the ompA gene has been present in 12 (75%) strains and the iss gene was present in 9 (56.25%) strains. Eight (50%) strains were found to present all three investigated genes. Conclusion: Presence of these genes is a strong indicatory to consider those strains as belonging to the APEC pathotype.

scholarly journals Abundance of pathogenic Escherichia coli, Salmonella typhimurium and Vibrio cholerae in Nkonkobe drinking water sources

2006 ◽  
Vol 4 (3) ◽  
pp. 289-296 ◽  
Author(s):  
Maggy N. B. Momba ◽  
Veronica K. Malakate ◽  
Jacques Theron
Keyword(s):  
Escherichia Coli ◽  
Drinking Water ◽  
Salmonella Typhimurium ◽  
Vibrio Cholerae ◽  
Water Samples ◽  
Pathogenic Bacteria ◽  
Culture Media ◽  
Water Sources ◽  
E Coli ◽  
Drinking Water Sources

In order to study the prevalence of enteric pathogens capable of causing infection and disease in the rural communities of Nkonkobe, bacterial isolates were collected from several surface water and groundwater sources used by the community for their daily water needs. By making use of selective culture media and the 20E API kit, presumptive Escherichia coli, Salmonella spp. and Vibrio cholerae isolates were obtained and then analysed by polymerase chain reaction assays (PCR). The PCR successfully amplified from water samples a fragment of E. coli uidA gene that codes for β-D-glucuronidase which is a highly specific characteristic of enteropathogenic E. coli, enterotoxigenic E. coli and entero-invasive E. coli. The PCR also amplified the epsM gene from water samples containing toxigenic V. cholerae. Although E. coli was mostly detected in groundwater sources, toxigenic V. cholerae was detected in both surface and groundwater sources. There was a possibility of Salmonella typhimurium in Ngqele and Dyamala borehole water samples. The presence of these pathogenic bacteria in the above drinking water sources may pose a serious health risk to consumers.

scholarly journals High diversity and variability of pipolins among a wide range of pathogenic Escherichia coli strains

2020 ◽  
Author(s):  
Saskia-Camille Flament-Simon ◽  
María de Toro ◽  
Liubov Chuprikova ◽  
Miguel Blanco ◽  
Juan Moreno-González ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pathogenic Bacteria ◽  
Phylogenetic Groups ◽  
Dna Viruses ◽  
E Coli ◽  
Host Interaction ◽  
Attachment Sites ◽  
Pathogenic Escherichia Coli ◽  
Wide Range ◽  
Recent Addition

AbstractSelf-synthesizing transposons are integrative mobile genetic elements (MGEs) that encode their own B-family DNA polymerase (PolB). Discovered a few years ago, they are proposed as key players in the evolution of several groups of DNA viruses and virus-host interaction machinery. Pipolins are the most recent addition to the group, are integrated in the genomes of bacteria from diverse phyla and also present as circular plasmids in mitochondria. Remarkably, pipolins-encoded PolBs are proficient DNA polymerases endowed with DNA priming capacity, hence the name, primer-independent PolB (piPolB).We have now surveyed the presence of pipolins in a collection of 2238 human and animal pathogenic Escherichia coli strains and found that, although detected in only 25 new isolates (1.1%), they are present in E. coli strains from a wide variety of pathotypes, serotypes, phylogenetic groups and sequence types. Overall, the pangenome of strains carrying pipolins is highly diverse, despite the fact that a considerable number of strains belongs to only three clonal complexes (CC10, CC23 and CC32). Comparative analysis with a set of 67 additional pipolin-harboring strains from GenBank further confirmed these results. The genetic structure of pipolins shows great flexibility and variability, with the piPolB gene and the attachment sites being the only common features. Most pipolins contain one or more recombinases that would be involved in excision/integration of the element in the same conserved tRNA gene. This mobilization mechanism might explain the apparent incompatibility of pipolins with other integrative MGEs such as integrons.In addition, analysis of cophylogeny between pipolins and pipolin-harboring strains showed a lack of congruence between several pipolins and their host strains, in agreement with horizontal transfer between hosts. Overall, these results indicate that pipolins can serve as a vehicle for genetic transfer among circulating E. coli and possibly also among other pathogenic bacteria.

Molecular Detection of wzx1 and wzy Genes in Multi Drugs Resistance E. coli Isolates

2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Mona Al-Terehi ◽  
Saba Saadoon Khazaal ◽  
Haidar J Muhammed ◽  
Russul Hikmat Behjet
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Gene Cluster ◽  
Molecular Detection ◽  
E Coli ◽  
Pcr Products ◽  
New Finding ◽  
O Antigens ◽  
Positive Results

Escherichia coli have been genetically changes in Iraqi environments, the present study was carried out to detection E coli isolates sero-group using o-antigens gene cluster, tow genes were amplified used multiplex PCR, the results of present study show that present isolates have more than one plasmid in different size (500-700 bp). PCR results show about 100% of isolates were had a positive results of PCR products of O45 wzx1 gene (451 bp) while 25% of isolates were a positive PCR products of O45 wzy1 gene (255 bp) and about 25% of isolates have tow genes. A new finding in present study that 50% of isolates have other copy of O45 wzx1 gene, these results concluded that it may be important in Iraqi isolates identification and classification however we need more information about genes sequencing.

Evaluation of Culture- and PCR-Based Detection Methods for Escherichia coli O157:H7 in Inoculated Ground Beef†

2005 ◽  
Vol 68 (8) ◽  
pp. 1566-1574 ◽  
Author(s):  
TERRANCE M. ARTHUR ◽  
JOSEPH M. BOSILEVAC ◽  
XIANGWU NOU ◽  
MOHAMMAD KOOHMARAIE
Keyword(s):  
Escherichia Coli ◽  
False Positive ◽  
False Negative ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
Detection Methods ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Perishable Product ◽  
Specificity And Sensitivity

Currently, several beef processors employ test-and-hold systems for increased quality control of ground beef. In such programs, each lot of product must be tested and found negative for Escherichia coli O157:H7 prior to release of the product into commerce. Optimization of three testing attributes (detection time, specificity, and sensitivity) is critical to the success of such strategies. Because ground beef is a highly perishable product, the testing methodology used must be as rapid as possible. The test also must have a low false-positive result rate so product is not needlessly discarded. False-negative results cannot be tolerated because they would allow contaminated product to be released and potentially cause disease. In this study, two culture-based and three PCR-based methods for detecting E. coli O157:H7 in ground beef were compared for their abilities to meet the above criteria. Ground beef samples were individually spiked with five genetically distinct strains of E. coli O157: H7 at concentrations of 17 and 1.7 CFU/65 g and then subjected to the various testing methodologies. There was no difference (P > 0.05) in the abilities of the PCR-based methods to detect E. coli O157:H7 inoculated in ground beef at 1.7 CFU/65 g. The culture-based systems detected more positive samples than did the PCR-based systems, but the detection times (21 to 48 h) were at least 9 h longer than those for the PCR-based methods (7.5 to 12 h). Ground beef samples were also spiked with potentially cross-reactive strains. The PCR-based systems that employed an immunomagnetic separation step prior to detection produced fewer false-positive results.

scholarly journals Characterization of a Hemoglobin Protease Secreted by the Pathogenic Escherichia coli Strain EB1

1998 ◽  
Vol 188 (6) ◽  
pp. 1091-1103 ◽  
Author(s):  
Ben R. Otto ◽  
Silvy J.M. van Dooren ◽  
Jan H. Nuijens ◽  
Joen Luirink ◽  
Bauke Oudega
Keyword(s):  
Escherichia Coli ◽  
Pathogenic Bacteria ◽  
Affinity Purification ◽  
Coli Strain ◽  
E Coli ◽  
Heme Acquisition ◽  
Pathogenic Escherichia Coli ◽  
Iga1 Protease ◽  
Animal Pathogens

Many pathogenic bacteria can use heme compounds as a source of iron. Pathogenic Escherichia coli strains are capable of using hemoglobin as an iron source. However, the mechanism of heme acquisition from hemoglobin is not understood for this microorganism. We present the first molecular characterization of a hemoglobin protease (Hbp) from a human pathogenic E. coli strain. The enzyme also appeared to be a heme-binding protein. Affinity purification of this bifunctional protein enabled us to identify the extracellular gene product, and to clone and analyze its gene. A purification procedure developed for Hbp allowed us to perform functional studies. The protein interacted with hemoglobin, degraded it and subsequently bound the released heme. These results suggest that the protein is involved in heme acquisition by this human pathogen. Hbp belongs to the so-called IgA1 protease-like proteins, as indicated by the kinetics of its membrane transfer and DNA sequence similarity. The gene of this protein appears to be located on the large pColV-K30 episome, that only has been isolated from human and animal pathogens. All these characteristics indicate that Hbp may be an important virulence factor that may play a significant role in the pathogenesis of E. coli infections.

Effect of microalgae growing on wastewater batch culture on escherichia coli and vibrio cholerae survival

1994 ◽  
Vol 30 (8) ◽  
pp. 295-302 ◽  
Author(s):  
N. Mezrioui ◽  
B. Oudra ◽  
K. Oufdou ◽  
L. Hassani ◽  
M. Loudiki ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Vibrio Cholerae ◽  
Batch Culture ◽  
Pathogenic Bacteria ◽  
Bacterial Load ◽  
Treated Wastewater ◽  
Coliform Bacteria ◽  
Organic Load ◽  
E Coli ◽  
Stabilization Ponds

The stabilization pond is one of the more important biological wastewater treatment systems, applied in many countries. An experiment treating wastewater by stabilization ponds under the arid climate of Marrakesh (Morocco) has been underway since 1985. The experimental installation, made from two lined stabilization ponds, received domestic sewage which carried not only the organic load but also a significant bacterial load and other microorganisms. In this new habitat, the cells' bacterial behaviour was affected by various physico-chemical and biological factors. It appears that in such treatment system, known for excessive algal production, the microalgae has evidently an influence on bacterial growth. In this paper, we proposed to appreciate how microalgae essentially: Chlorella (Chlorophyta), Synechococcus andSynechocystis (Cyanobacteria), can affect the behaviour, survival and temporal evolution of Escherichia coli and Vibrio cholerae. In wastewater stabilization ponds of Marrakesh high levels of V. cholerae and low concentrations of coliform bacteria were noted during summer periods. This period coincided with a bloom of picocyanobacteria associated with a weak relative abundance of Chlorella. Some interactions tests were carried out with these bacteria and these algae, using a treated wastewater batch culture. Results show that the green algae reduces V. cholerae (pathogenic bacteria) abundances more than E. coli (fecal contamination bacteria) where as better survival of this pathogenic bacteria was noted in presence of Cyanobacteria. The die-off of E. coli appears to be more reduced in presence of Cyanobacteria than Chlorella. Furthermore, the alkaline pH seems to present a more bactericidal effect on E. coli than on V. cholerae. Thus, the Cyanobacteria blooms, associated with a weak percentage of Chlorella abundance, occurring periodically during summer in sewage stabilization ponds of Marrakesh, will be considered as one of the major factors leading to high levels of V. cholerae and low abundances of fecal coliform bacteria during the hot period.

scholarly journals Reverse Transcription-Multiplex PCR Assay for Simultaneous Detection of Escherichia coli O157:H7, Vibrio cholerae O1, and Salmonella Typhi

2004 ◽  
Vol 50 (11) ◽  
pp. 2037-2044 ◽  
Author(s):  
Nicole J Morin ◽  
Zhilong Gong ◽  
Xing-Fang Li
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Vibrio Cholerae ◽  
Reverse Transcription ◽  
Simultaneous Detection ◽  
Salmonella Typhi ◽  
Escherichia Coli O157 ◽  
Single Tube ◽  
E Coli ◽  
Vibrio Cholerae O1

Abstract Background: Escherichia coli O157:H7, Vibrio cholerae O1, and Salmonella Typhi are pathogenic bacteria that can be found in contaminated water supplies throughout the world. No currently available assays can simultaneously detect and identify all three pathogens. Our aim was to develop a rapid and reliable technique for simultaneous detection of these pathogens. Methods: Four unique genes were chosen as the targets of detection. Forward and reverse primers were designed to specifically amplify different sizes of these target genes: a 239-bp region of the E. coli O157 lipopolysaccharide (LPS) gene (rfbE); a 179-bp region of the H7 flagellin gene (fliC); a 419-bp region of the V. cholerae O1 LPS gene (rfbE); and a 329-bp region of Salmonella Typhi LPS gene (tyv). To ensure the detection of only viable replicating bacteria, RNA was extracted for analysis. After reverse transcription, cDNAs were simultaneously amplified in a single tube by multiplex PCR. The multiplex PCR products were analyzed by gel electrophoresis. To characterize the assay we analyzed, in a blinded fashion, seven unknown RNA samples containing various combinations of total RNA from these bacteria as well as clinical isolates. Results: All seven unknown RNA samples were correctly identified. The assay was able to detect and identify as few as 30 cells of E. coli O157:H7 and Salmonella Typhi in clinical isolates, and the presence of other bacteria did not interfere with the analysis. Conclusion: An assay combining reverse transcription with single-tube multiplex PCR was successfully developed and validated for simultaneous detection of viable E. coli O157:H7, V. cholerae O1, and Salmonella Typhi.

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